¹⁵N NMR Hyperpolarization of Radiosensitizing Antibiotic Nimorazole via Reversible Parahydrogen Exchange in Microtesla Magnetic Fields

Abstract

Nimorazole belongs to the imidazole-based family of antibiotics to fight against anaerobic bacteria. Moreover, nimorazole is now in Phase 3 clinical trial in Europe for potential use as a hypoxia radiosensitizer for treatment of head and neck cancers. We envision the use of [¹⁵N₃]nimorazole as a theragnostic hypoxia contrast agent that can be potentially deployed in the next-generation MRI-LINAC systems. Here, we report the first but very important steps to create long-lasting (for tens of minutes) hyperpolarized state on three ¹⁵N sites of [¹⁵N₃]nimorazole with T₁ of up to ~6 minutes. The nuclear spin polarization was boosted by ~67000-fold at 1.4 T (corresponding to P_15N of 3.2%) via ¹⁵N-¹⁵N spin-relayed SABRE-SHEATH hyperpolarization technique relying on simultaneous exchange of [¹⁵N₃]nimorazole and parahydrogen on polarization transfer Ir-IMes catalyst. The isomeric side product of [¹⁵N₃]nimorazole synthesis was hyperpolarized too. The presented results pave the way to efficient spin-relayed SABRE-SHEATH hyperpolarization of a wide range of imidazole-based antibiotics and chemotherapeutics.

Graphical Abstract

Synthesis of ¹⁵N₃-enriched antibiotic and hypoxic radiosensitizer nimorazole is reported. Spin-relayed Signal Amplification By Reversible Exchange in microtesla magnetic field yielded up to 3.2% ¹⁵N nuclear spin polarization with relaxation time constant of 5.9 ± 0.2 min at 1.4 T. The isomer of nimorazole formed in the synthesis was also hyperpolarized indicating that this approach can be extended to a wide range of nitroimidazole-based drugs.

Introduction

Equilibrium nuclear spin polarization (P) of key biologically-relevant nuclei such as ¹H, ¹³C and ¹⁵N is on the order of 10⁻⁶-10⁻⁵ at clinically relevant conditions of body temperature and the magnetic field of up to 3 T. Since signal-to-noise ratio (SNR) is directly proportional to P in magnetic resonance (MR), NMR techniques including most notably MRI have inherently low detection sensitivity. As a result, clinical MRI is typically limited to imaging of protons of water and lipids, which have high physiological concentrations of tens of moles/L. In contrast, MR imaging of dilute metabolites is a grand challenge despite potential benefits of providing rich metabolic information, which is central to diagnosis of diseases with aberrant metabolism such as cancer, diabetes, etc. NMR hyperpolarization techniques allow transiently increasing P up to the order of unity.^{[1, 2]} This massive increase in sensitivity gains by 4–5 orders of magnitude can be employed for metabolic imaging of hyperpolarized (HP) compounds, which are typically injected and act as metabolic contrast agents.^{[3–5] 13}C-HP [1-¹³C]pyruvate is the most notable metabolite that has been developed so far to probe upregulated glycolysis in cancer and many other diseases.^[6–11] Numerous clinical trials are now enabled because access to this HP contrast agent became possible via dissolution Dynamic Nuclear Polarization (d-DNP).^[12–14] Despite many technology breakthroughs^[15–17] over the recent years, d-DNP has many shortcomings of high equipment- (>$2M) and operational costs, and long polarization times.^[12] For example, hyperpolarization of ¹⁵N sites has been a main challenge for conventional d-DNP requiring custom ¹H→¹⁵N cross-polarization hardware to achieve efficient hyperpolarization with P_15N>15%.^[18] A number of alternative hyperpolarization techniques are now under development to address d-DNP limitations.^{[1, 19]}

Signal Amplification by Reversible Exchange (SABRE)^[20–22] technique relies on the simultaneous chemical exchange of parahydrogen (p-H₂) and to-be-hyperpolarized biomolecule on a hexacoordinate Ir complex, usually possessing IMes or a similar ligand.^[23] At the appropriate magnetic field, nuclear spin order of parahydrogen-derived protons is transferred to the nuclei of coordinated substrate. Subsequent dissociation of the complex liberates HP biomolecule to the solution. SABRE technique underwent a rapid development over the past few years,^{[19, 24]} and the list of amenable biomolecules has been rapidly expanding.^[25–30] Although [1-¹³C]pyruvate and other ¹³C-containing biomolecules are biomedically interesting, their primary limitation is a relatively short lifetime of HP state with exponential decay time constant T₁ on the order of 1 min in most favorable cases.^{[4, 5, 12]} HP ¹⁵N sites have garnered a lot of attention recently because their T₁ can be on the order of 10 min or longer ^{[28, 31–33]} – as a result, the detection window of HP injectable biomolecules can be significantly expanded.^{[34, 35]}

Although there have been some reports for hyperpolarization of ¹⁵N-labeled biomolecules via d-DNP, this technology is not well suited due to ultra-long polarization times of several hours, and low polarization yields on top of the other d-DNP limitations.^{[35, 36]} As a result, the progress of biomedical translation of HP ¹⁵N has been relatively slow.

A variant of SABRE technique called SABRE-SHEATH (SABRE in SHield Enables Alignment Transfer to Heteronuclei, Figure 1)^{[37, 38]} allows for very efficient polarization of ¹⁵N sites in ¹⁵N-containing compounds with fast polarization times (1 min or less) and high yields of P_15N > 30%.^[39] In this technique, microtesla magnetic fields are employed to promote polarization transfer from p-H₂ derived protons to heteronuclei. SABRE-SHEATH was shown to hyperpolarize metronidazole,^[40] the FDA-approved antibiotic that can be safely administered in large doses of up to several grams.^{[41, 42]} This antibiotic is structurally similar to many nitroimidazole-based ¹⁸F-labeled radiotracers, employed for hypoxia imaging in cancer and other diseases.^[43–45] More recently, we have demonstrated that spin-relays^{[46, 47]} can provide efficient polarization of ¹⁵N-¹⁵N spin-spin coupled sites in metronidazole with −¹⁵NO₂ group gaining high levels of polarization (>16%) and very long T₁ of ~10 min.^[48] The −NO₂ group undergoes reduction during hypoxia sensing process, but ¹⁸F-labeled radiotracers are not capable of distinguishing metabolized compounds, and radiotracer’s clearance from surrounding tissues takes several hours.^[49] As a result, clearance time of ~2 h is typically required prior to nitroimidazole-based positron emission tomography (PET) scan to gain sufficient contrast with hypoxic region. ¹⁵N-HP nitroimidazoles can potentially mitigate this shortcoming, and enable fast hypoxia scan (<5 min) without ionizing radiation.

Figure 1.

Schematics of spin-relayed^[46] SABRE-SHEATH^{[37, 38] 15}N hyperpolarization of [¹⁵N₃]nimorazole. Both p-H₂ and [¹⁵N₃]nimorazole are in chemical exchange with the Ir complex. In the microtesla magnetic field, hyperpolarization is transferred from p-H₂-derived hydride protons to the ¹⁵N nuclei of coordinated substrate via spin-relayed mechanism. Dissociation of HP [¹⁵N₃]nimorazole from the Ir complex allows to obtain free HP [¹⁵N₃]nimorazole in solution.

Here, we report on synthesis and SABRE-SHEATH hyperpolarization study of [¹⁵N₃]nimorazole. Although it has been originally developed as an antibiotic against anaerobic infections,^[41] it is currently under a Phase 3 clinical study in Europe as a radio-sensitizing agent for the treatment of head and neck cancer.^{[50, 51]} If successful, nimorazole will be integrated in the first-line of cancer chemotherapy treatment. We envision that HP [¹⁵N₃]nimorazole can be simultaneously employed for both tumor radiosensitization and also as an imaging agent to report on tumor hypoxia status.

Results and Discussion

Typical SABRE-SHEATH experiments employed ~600 μL of solution containing ~100 mM of substrate and ~5 mM of SABRE pre-catalyst [Ir(IMes)(COD)]Cl^{[23, 53]} in CD₃OD, placed in a medium-wall 5 mm NMR tube tightly connected with 1/4 inch outer-diameter Teflon tube. NMR tube was placed into the flask filled with water to maintain the constant temperature, and the flask itself was placed in the MuMETAL magnetic shield with the ability to control magnetic field inside via an additional solenoid magnet. The SABRE catalyst was activated via bubbling of p-H₂ gas through the solution at 20 standard cubic centimeters per minute (sccm) flow rate and 7.8 bar pressure for ~2 h. Typical SABRE-SHEATH experiments were performed using the following procedure. p-H₂ gas was bubbled through the solution at 70 sccm flow rate and 7.8 bar pressure for 60 s. Then gas flow through the solution was terminated by opening the valve on the by-pass line (Scheme 2). NMR tube was pulled out from the magnetic shield, wiped with a paper towel and placed into the probe of 1.4 T Nanalysis NMReady-60PRO bench-top NMR spectrometer for ¹⁵N NMR detection.

Scheme 2.

Scheme of experimental setup.

Our initial ¹⁵N SABRE-SHEATH experiments using nimorazole with natural abundance of ¹⁵N nuclei were unsuccessful, and ¹H detection of SABRE-HP samples yielded very small ¹H signal enhancements of ca. 3–4-fold (Figure S1). Therefore, we performed the synthesis of isotopically labeled [¹⁵N₃]nimorazole to increase ¹⁵N enrichment from ~0.4% (natural abundance) to ~99% (Scheme 1). In the first step, [¹⁵N₂]imidazole was prepared with 50–60% yield via condensation of glyoxal, formaldehyde and ¹⁵NH₄Cl used as a source of ¹⁵N isotope enrichment.^[52] Subsequent nitration with H¹⁵NO₃/H₂SO₄ allowed to obtain 4(5)-[¹⁵N]nitro-[1,3-¹⁵N₂]imidazole with 30% yield. Next, this compound was alkylated with 4-(2-chloroethyl)morpholine in the presence of K₂CO₃. Due to tautomerism of the imidazole heterocycle, both nitrogen atoms possess nucleophilic properties, leading to unselective alkylation. Thus, the yield of [¹⁵N₃]nimorazole was only 20% while the yield of its isomer (here termed [¹⁵N₃]isonimorazole for convenience) was 60% in the last step. The prevalence of isonimorazole is not unexpected since the corresponding nitrogen atom is more nucleophilic than the other one because it is further away from electron accepting nitro group. Moreover, steric hindrance effects favor the formation of isonimorazole compared to nimorazole. The two ¹⁵N-labeled isomers were separated chromatographically. See SI for additional synthetic information and spectral characterization of the products.

Scheme 1.

Synthesis of [¹⁵N₃]nimorazole (4-(2-(5-[¹⁵N]nitro-1H-[1,3-¹⁵N₂]imidazol-1-yl)ethyl)morpholine) and side product [¹⁵N₃]isonimorazole (4-(2-(4-[¹⁵N]nitro-1H-[1,3-¹⁵N₂]imidazol-1-yl)ethyl)morpholine). Blue and red colors denote the sources of ¹⁵N isotope label.

SABRE-SHEATH experiments using [¹⁵N₃]nimorazole demonstrated efficient ¹⁵N hyperpolarization on all three ¹⁵N nuclei—the representative ¹⁵N NMR spectrum is shown in Figure 2a. The finding that all three ¹⁵N sites have nearly equal %P_15N (ranging from 1.9% to 3.2%) is similar to the recent study of spin-relayed^{[46, 47]} (via the network formed by ²J_15N-15N) polarization transfer in [¹⁵N₃]metronidazole.^[48] Although the maximum %P_15N level for [¹⁵N₃]nimorazole was lower than that for [¹⁵N₃]metronidazole by a factor of ~5.3 (3.2% vs. 17%^[48]), the concentration of to-be-hyperpolarized substrate (and catalyst) was ~4.2-fold greater in [¹⁵N₃]nimorazole studies. Moreover, a somewhat higher hyperpolarization efficiency of [¹⁵N₃]metronidazole may also be tentatively attributed to the electron donating properties of methyl group (lacking in [¹⁵N₃]nimorazole heterocycle) likely modulating the efficiency of SABRE-SHEATH polarization transfer process. We conclude that ¹⁵N-¹⁵N spin-relayed polarization transfer is a relatively general phenomenon, which is now documented in the molecular motifs of [¹⁵N₃]nimorazole, [¹⁵N₃]metronidazole and [¹⁵N₃]isonimorazole (see below). In this spin-relayed mechanism,^{[48] 15}N-3 nuclear spin is hyperpolarized first via polarization transfer from p-H₂-derived hydrides (Figure 1). Second, polarization is transferred from ¹⁵N-3 to ¹⁵N-1 via ²J_15N-15N. Finally, polarization is transferred from ¹⁵N-1 to ¹⁵NO₂ via another ²J_15N-15N (Figure 1).^[48]

Figure 2.

(a) Single-scan ¹⁵N NMR spectrum of HP [¹⁵N₃]nimorazole. Conditions: ~140 mM substrate, ~7 mM catalyst, 52 °C, 7.8 bar p-H₂ pressure, 70 sccm gas flow rate, 0.4 μT magnetic field inside the magnetic shield. (b) ¹⁵N NMR spectrum of thermally polarized neat [¹⁵N]pyridine acquired with 16 signal accumulations. Note this spectrum intensity is divided by the number of signal accumulations and scaled by a factor of 128. The spectra were acquired on a 1.4 T bench-top NMR spectrometer. The ¹⁵N NMR spectrum of HP [¹⁵N₃]nimorazole recorded on a 7.05 T NMR spectrometer is presented in Figure S2.

To maximize the polarization levels, >2 h of activation of SABRE complex^{[38, 55]} in the presence of [¹⁵N₃]nimorazole is required (Figure 3a). This activation time is comparable to that observed with [¹⁵N₃]metronidazole under similar conditions.^[56] The magnetic field profile of polarization transfer in microtesla field range exhibits a clear maximum at ~0.4 μT for all three ¹⁵N sites (Figure 3b). The maximum position is the same as that for [¹⁵N₃]metronidazole,^[48] further supporting the similarity in J coupling network between the polarizing ¹⁵N spins in these two molecules. Moreover, the microtesla magnetic field profile is similar for all three ¹⁵N nuclei of [¹⁵N₃]nimorazole, serving as an additional proof of spin-relayed polarization transfer mechanism discussed above. Furthermore, the dynamics of ¹⁵N polarization buildup showed that characteristic exponential buildup times are similar (T_b = 23–29 s) for the three ¹⁵N nuclei (Figure 3c) at optimal B_T of ~0.4 μT. Note when sub-optimal B_T is employed (e.g., <0.04 μT as in Figure S3a), the equilibrium %P_15N is achieved faster (T_b = 3.1–3.5 s) albeit yielding much lower %P_15N by at least several folds.

Figure 3.

SABRE-SHEATH hyperpolarization of [¹⁵N₃]nimorazole at ~5 mM catalyst and ~100 mM substrate initial concentrations: (a) SABRE-SHEATH catalyst activation kinetics in the presence of [¹⁵N₃]nimorazole at 20 °C, 7.8 bar p-H₂ pressure, 20 sccm gas low rate and 0.4 μT. (b) SABRE-SHEATH magnetic field profile of [¹⁵N₃]nimorazole obtained at 20 °C, 7.8 bar p-H₂ pressure and 70 sccm p-H₂ flow rate. (c) SABRE-SHEATH polarization buildup of [¹⁵N₃]nimorazole obtained at 20 °C, 7.8 bar p-H₂ pressure, 70 sccm gas low rate and 0.4 μT. (d) Relaxation kinetics of HP [¹⁵N₃]nimorazole at 0.4 μT. (e) Relaxation kinetics of HP [¹⁵N₃]nimorazole at ~10 μT. (f) Relaxation kinetics of HP [¹⁵N₃]nimorazole at 1.4 T. (g) The gas flow rate dependence of SABRE-SHEATH polarization of [¹⁵N₃]nimorazole nitro group obtained at different pressures, 20 °C and 0.4 μT. The inset shows pressure dependence of nitro group ¹⁵N polarization obtained at 99 sccm gas flow rate. (h) Temperature dependence of SABRE-SHEATH polarization of [¹⁵N₃]nimorazole obtained at 7.8 bar p-H₂ pressure, 70 sccm gas low rate and 0.4 μT.

Next, the ¹⁵N T₁ relaxation dynamics of HP [¹⁵N₃]nimorazole was studied at three relevant static magnetic fields. At ~0.4 μT (the optimal polarization transfer field for [¹⁵N₃]nimorazole), the signals of three ¹⁵N nuclei decayed with approximately the same characteristic relaxation time T₁ = 29–33 s (Figure 3d). This ¹⁵N T₁ clustering at ~0.4 μT (and similar type of clustering at ~10 μT in Figure 3e, and < 0.04 μT in Figure S3b) is important for two reasons. First, it is consistent with the fact that these three ¹⁵N nuclei are strongly coupled at microtesla magnetic fields and thus continuously share magnetization with each other via ¹⁵N-¹⁵N spin relays.^{[47, 48]} Second and more importantly, our recent study of [¹⁵N₃] and [¹⁵N₂]metronidazole isotopologues clearly demonstrated the detrimental role of ¹⁴N quadrupolar nucleus presence in the spin-relayed network.^[56] In this case, two-bond ¹⁵N-¹⁴N spin-spin coupling results in a significantly lower effective ¹⁵N T₁ for all (remaining) ¹⁵N sites in [¹⁵N₂]metronidazole versus [¹⁵N₃]metronidazole: ¹⁵N T₁ clustering of 8–9 seconds versus 23–31 seconds respectively under similar (1:20) catalyst-to-substrate ratio as the one studied here.^[56] [¹⁵N₃]nimorazole T₁ relaxation cluster of 29–33 seconds (Figure 3d) overlaps well with [¹⁵N₃]metronidazole cluster^[56] rather than with [¹⁵N₂]metronidazole. We rationalize this very important observation in a way that while two-bond ¹⁵N-¹⁴N spin-spin coupling results in efficient ¹⁴N-rendered quadrupolar depolarization effects, the corresponding three-bond coupling has a negligible effect. This is advantageous in the context of development of ¹⁵N HP contrast agents with SABRE-SHEATH approach as remote N-sites don’t require ¹⁵N isotopic labeling to maximize polarization efficiency of the ¹⁵N spins.

Once SABRE-SHEATH polarization transfer was completed, the samples were transferred from the microtesla field to the 1.4 T bench-top NMR spectrometer through the Earth’s magnetic field. In this experimental protocol, relaxation of ¹⁵N nuclei of [¹⁵N₃]nimorazole at the Earth’s field (ca. 10 μT in our lab, apparently due to presence of various magnetic equipment etc.) may have an impact on the resultant ¹⁵N polarization levels detected at 1.4 T due to T₁ relaxation process during the sample transfer (Figure 3e). At the clinically relevant field of 1.4 T of bench-top NMR spectrometer, ¹⁵N T₁ values for the three ¹⁵N sites were significantly lengthened to 51 ± 4 s for ¹⁵N-3, 109 ± 4 s for ¹⁵N-1 and 353 ± 13 s for ¹⁵NO₂ sites, respectively. Although slower relaxation for the ¹⁵N nuclei of ¹⁵NO₂ is expected due to relative isolation of this ¹⁵N site from any protons, this long T₁ of nearly 6 min is remarkable as it allows storing ¹⁵N polarization for tens of minutes (Figure 3f).

Parahydrogen flow rate dependence of ¹⁵N SABRE-SHEATH hyperpolarization of [¹⁵N₃]nimorazole demonstrated that polarization plateaus after ~50 sccm gas flow rate (Figure 3g). Increase in p-H₂ pressure leads to a nearly linear %P_15N increase (Figure 3g) indicating that %P_15N can be further enhanced at elevated p-H₂ partial pressure.^[55] The temperature dependence of [¹⁵N₃]nimorazole polarization exhibits a clear maximum at ~54 °C – with %P_15N being an order of magnitude greater than that at ~20 °C. Thus, temperature optimization is crucial for efficient SABRE-SHEATH hyperpolarization of [¹⁵N₃]nimorazole. This observation is different from the optimal temperature of nearly all other SABRE-SHEATH substrates studied to date with optimal %P_15N achieved at room temperature of ~20 °C^{[25, 27, 38, 40]} with the exception of [¹⁵N₂]imidazole^[57] (t_OPT > 75 °C)—this is likely explained by the different kinetics of nimorazole dissociation from Ir complex.

Similar measurements were carried out at lower concentrations (1 mM catalyst and 20 mM [¹⁵N₃]nimorazole, Figure S4), and this different concentration regime rendered overall similar results. The addition of co-substrate (pyridine^[58]) to the initial solution promoted the rate of SABRE complex activation (Figure S5). However, the maximum in the magnetic field profile of SABRE-SHEATH polarization shifted from ~0.4 μT to ~0.15–0.20 μT, indicating that the active complex likely contained both pyridine and [¹⁵N₃]nimorazole molecules. Characteristic polarization buildup and relaxation times at microtesla magnetic field were similar to those observed without a co-substrate (compare Figure 3 and Figure S5). On the other hand, ¹⁵N relaxation at the Earth’s magnetic field was more efficient, while relaxation at 1.4 T was slower in the presence of pyridine (Figure 3 and Figure S5). Taken altogether, addition of pyridine as a co-substrate led to significant %P_15N decrease by 2–6-fold.

We have also explored the possibility to hyperpolarize the isomer of [¹⁵N₃]nimorazole ([¹⁵N₃]isonimorazole) using SABRE-SHEATH. In this compound, the nitro group is located closer to the donor N atom that can coordinate to Ir complex than in nimorazole and metronidazole. This proximity of nitro group is expected to complicate coordination of the substrate to the metal center due to both steric and electronic effects.^{[54, 59]} Nevertheless, we have demonstrated that SABRE-SHEATH hyperpolarization of all ¹⁵N nuclei of [¹⁵N₃]isonimorazole is possible, resulting in up to 0.47% polarization (Figure S6). This observation paves the way to SABRE-SHEATH hyperpolarization of other biologically active nitroimidazole derivatives containing nitro group in the α position to the nitrogen atom with a lone electron pair. This range of compounds includes prospective antitumor prodrug evofosfamide (a.k.a. TH-302^[60]), antibiotics azomycin and benznidazole, immunosuppressive drug azathioprine and many others.

Dependence of SABRE-SHEATH polarization efficiency of [¹⁵N₃]isonimorazole on magnetic field, polarization buildup time and temperature was investigated, as well as relaxation of ¹⁵N hyperpolarization at three different magnetic fields. Magnetic field profile demonstrated the maximum at 0.25 μT (Figure S8a), which is lower than corresponding maximum for [¹⁵N₃]nimorazole. Characteristic polarization buildup time for [¹⁵N₃]isonimorazole (Figure S8b) was also lower than for [¹⁵N₃]nimorazole (~12–13 s vs. 23–29 s). Temperature dependence of SABRE-SHEATH polarization for [¹⁵N₃]isonimorazole was also dramatically different: the broad maximum was observed at ~30–43 °C (Figure S8f), in contrast to a sharper peak at ~54 °C for [¹⁵N₃]nimorazole. Comparing the efficiency of hyperpolarization for the three ¹⁵N sites, ¹⁵NO₂ group exhibited the highest polarization levels, similar to [¹⁵N₃]nimorazole. However, in case of [¹⁵N₃]isonimorazole two ¹⁵N nuclei in imidazole ring exhibited similar polarization levels, while for [¹⁵N₃]nimorazole ¹⁵N-1 site was polarized more efficiently.

Relaxation of ¹⁵N nuclei in [¹⁵N₃]isonimorazole at microtesla and the Earth’s magnetic field was faster than in [¹⁵N₃]nimorazole. Similar values (19–21 s and ~18–24 s) were observed at both magnetic fields under study (Figures S8c and S8d). On the other hand, at 1.4 T relaxation of [¹⁵N₃]isonimorazole ¹⁵N nuclei was slower compared to [¹⁵N₃]nimorazole (Figure S8e): 90 ± 10 s for ¹⁵N-3 site, 128 ± 14 s for ¹⁵N-1 site and 417 ± 27 s for ¹⁵NO₂ group, respectively. The long relaxation time for ¹⁵NO₂ group of [¹⁵N₃]isonimorazole signifies that other nitroimidazole compounds yet to be hyperpolarized by SABRE will likely exhibit a similarly long relaxation times of HP ¹⁵N sites.

The feasibility of 2D sub-second ¹⁵N MRI visualization of HP [¹⁵N₃]nimorazole was demonstrated. Figure 4 presents 2D ¹⁵N TrueFISP MR images of a 5 mm NMR tube containing ~110 mM HP [¹⁵N₃]nimorazole acquired on a 9.4 T MRI scanner. Note the very high spatial (0.5×0.5 mm²/pixel) and temporal (356 ms) resolution that can be potentially achievable with HP ¹⁵N MRI of [¹⁵N₃]nimorazole – first two images in each series are shown for two different projection views. This proof-of-principle demonstration opens the prospects for utilization of SABRE ¹⁵N hyperpolarization of [¹⁵N₃]nimorazole in bioimaging applications such as reporting on tumor hypoxia status in a manner similar to that of nitroimidazole-based PET tracers.^[43–45]

Figure 4.

¹⁵N TrueFISP MRI of HP [¹⁵N₃]nimorazole (~110 mM) in a 5 mm NMR tube using multi-nuclear 9.4 T MRI scanner. Frequency offset was adjusted to the signal of ¹⁵NO₂ group. The imaging parameters: spectral width (SW) = 5 kHz, repetition time (TR) = 14 ms, echo time (TE) = 7 ms, flip angle = 30˚. (a–b) Axial projection (the first and the second scan): scan acquisition time = 356 ms, matrix size = 32×32 (zero-filled to 512×512), FOV = 1.6 cm × 1.6 cm, spatial resolution = 0.5×0.5 mm²/pixel. (c–d) Coronal projection (the first and the second scan): scan acquisition time = 684 ms, matrix size = 64×64 (zero-filled to 512×512), FOV = 7.8 cm × 7.8 cm, spatial resolution = 1.2×1.2 mm²/pixel. Full sets of 16 consecutively recorded MR images are presented in Figure S9 (axial projection) and Figure S10 (coronal projection). ¹⁵N NMR spectra acquired at the similar conditions demonstrated P_15N of 0.78% for the ¹⁵NO₂ group and P_15N of 0.33% for the ¹⁵N-3 site (Figure S11).

Biomedical Outlook

The reported herein study was performed in CD₃OD because it is readily available and it has been employed by the vast majority of published in vitro studies. For future in vivo applications, we envision hyperpolarization in a more biocompatible ethanol solvent (SABRE hyperpolarization of nicotinamide in ethanol has been well documented^[55]) followed by rapid catalyst removal using functionalized resins^{[39, 61]} and reconstitution to aqueous medium via rapid vacuum distillation of ethanol down to less that 10% content. These two steps should take less than 0.5 minute based on our experience resulting in relatively small polarization losses for all three ¹⁵N sites. The presence of 10% ethanol is a generally accepted clinical practice in the context of PET tracers’ administration with up to 20 mL dose.^[62] The development of bio-injectable catalyst-free formulation is the next key step for SABRE-SHEATH hyperpolarization technique.

Although catalyst activation takes ~2 h, it can be performed well in advance of the hyperpolarization procedure with normal hydrogen (as the materials are stable for many hours), and the bolus of [¹⁵N₃]nimorazole can be hyperpolarized in ~1 min. Although the optimum polarization temperature is ~50–60 °C (Figure 3h), this is not a translational challenge—our previous experience with other parahydrogen-hyperpolarized ¹³C compounds produced at 68 °C shows that the HP liquid equilibrates to below 37 °C at the time of the actual in vivo injection.^{[63, 64]} We note that the produced dose of HP [¹⁵N₃]nimorazole in NMR tube of ~50 μmoles is certainly sufficient for future pilot in vivo studies in small rodents—previous successful studies with HP ¹³C injectable agents employed ~5 μmole dose in mice.^{[9, 64]} We envision that the injected nimorazole will undergo cellular uptake within one minute based on the pharmacodynamics data for structurally similar nitroimidazole compounds.^[45] The subsequent metabolism of nitroimidazole compounds in hypoxic cells leads to formation of nitroso and hydroxylamine intermediates and amino-derivative metabolic products, Figure 5.^[65] The ab initio calculations of ¹⁵N chemical shifts using Gaussian’09 (see SI for details) for nimorazole and its metabolic reduction products reveal that the associated ¹⁵N chemical shifts of all labelled ¹⁵N sites are very sensitive to reduction process. While the nitro-group nitrogen is the most sensitive one (with nearly 800 ppm dynamic range), it may not necessarily be the most useful sensing site for hydroxylamino- and amino-derivatives because of the anticipated decrease in ¹⁵N T₁ due to directly attached protons. On the other hand, while the dynamic range of the other two sites is less dramatic (12 ppm and 20 ppm respectively), these two ¹⁵N sites will likely retain their long T₁ in vivo. To summarize, the relaxation properties and the ¹⁵N chemical shift dispersion of metabolic products bode well for future in vivo studies of using HP [¹⁵N₃]nimorazole as an injectable hypoxia sensor.

Figure 5.

Metabolism of nitroimidazole compounds on the example of nimorazole in hypoxic environment.^[65] Color-coded values of ¹⁵N chemical shifts (in ppm) were computed for aqueous media using Gaussian’09 ab initio calculations (see SI for details).

Conclusion

To summarize, we report on efficient synthesis and SABRE-SHEATH hyperpolarization of ¹⁵N-labeled [¹⁵N₃]nimorazole. The HP state with %P_15N of up to 3.2% ¹⁵N polarization is created on all three ¹⁵N sites, and it persists for tens of minutes with ¹⁵N T₁ decay of 5.9 ± 0.2 min at clinically relevant field of 1.4 T. This report paves the way to future theragnostic imaging applications of [¹⁵N₃]nimorazole and other HP nitroimidazoles as cancer chemotherapeutic agents and as tumor hypoxia sensors that may significantly aid to advance personalized care of cancer patients.

The relaxation dynamics results presented here provide valuable insights for the rational development of schemes and molecular substrates for efficient spin-relayed ¹⁵N SABRE-SHEATH hyperpolarization. Although synthesis yields a side product, we were able to successfully separate and purify both compounds. The feasibility of SABRE-SHEATH ¹⁵N hyperpolarization of the synthesized isomer of [¹⁵N₃]nimorazole with ¹⁵NO₂ group in α position to donor nitrogen atom of imidazole ring opens opportunities for efficient hyperpolarization of a wide range of other biomedically interesting molecules with very long-lived HP state retained by NO₂ group. For example, HP azomycin can potentially act as a suitable pH sensor (pKa ~ 7.0),^[57] and TH-302^[66] can be employed as a theragnostic agent for cytotoxic chemotherapeutics selectively activating on hypoxic tumors, which are typically difficult to treat using conventional chemotherapies.